+ was, however, not affected by the presence of the activator. Thus, it would seem impossible at the present time to offer a rational explana
tion, in terms of electrokinetic potentials, of the activation and inhibition of plant phospholipase D activity. Obviously, the particulate enzyme activity is controlled by different physicochemical conditions from those operating in the case of the soluble enzyme, and much more needs to be known about the behavior of ion-colloid interactions in a diethyl ether medium.
II. PHOSPHOLIPID SYNTHESIS
Our current knowledge of the synthesis of phospholipids in living tissue has been reviewed recently by Rossiter (26). In Fig. 6 is a summary of the reactions concerned in the synthesis of phosphatidylcholine, phospha
tidylethanolamine, and phosphatidylinositol (monophosphoinositide). Less certain is the step leading to the plasmalogens (phosphatidalcholine and phosphatidalethanolamine). Figure 7 gives the series of reactions leading to sphingomyelin. Two outstanding features of these systems are the central position occupied by phosphatidic acid (and possibly its plas-malogen analogue) in the synthesis of the phosphoglycerides and the importance of the cytidine coenzymes in the introduction of the bases into the intact phospholipids. Practically all these reactions have been studied with tissue homogenates or cell particle preparations, and it would seem that phospholipid synthesis is carried out in both the mitochondria and the endoplasmic reticulum (microsomes). Thus, these important cellular structures both rich in phospholipids may both synthesize their own phospholipid. Each step will be discussed separately, as numbered in Figs. 6 and 7.
1. GLYCEROKINASE
Bublitz and Kennedy (4) studied a purified soluble glycerokinase from rat liver. The activity was stabilized by glycerol, cysteine, and E D T A added to the stored solutions. Various sulfhydryl compounds stimulated the activity, and preincubation of the enzyme with —SH-reacting sub
stances such as iodoacetamide and p-chloromercuribenzenesulfonate pro
duced considerable inhibition. Magnesium at a concentration of 0.003 M was necessary for optimum activity, but higher concentrations were dis
tinctly inhibitory. The magnesium could be replaced by manganese, but calcium was inhibitory. The enzyme can transfer phosphate from both A T P and U T P to glycerol, and in both cases the reaction is inhibited by added ADP, suggesting product inhibition of the reaction.
Phosphoryl
choline CDP. choline
Cytidine diphosphate diglyceride
" Inositol
•CMP
(10)
G
( 9 )Phosphatidyl-inositol
(Monophosphoinositide)
GLYCEROL (1) \ A D P L-α- Gly cerophosphate
^oA L-α-Phosphatidic acid
(3) M P i ^-1 D-a, β-Diglyceride
(4)
j^CMP.
L-α-Lecithin
(or phosphatidyethanolamine) FI G . 6. Reactions involved in the biosynthesis of the phosphoglycerides.
FATTY ACID (5) /"ATP +
VAMP + Fatty acyl CoA
ATP + CoA PPi
Choline (or ethanolamine) (6) ATP
AD Ρ Phosphorylcholine
(7) j V P P i
• Cytidine diphosphate choline (or ethanolamine)
(8) Plasmalogen diglyceride Plasmalogen + CMP
198 J. Β. DAVENPORT
5. PHOSPHOLIPID S 199
2. ACYLATIO N O F L-ÛÉ-GLYCEROPHOSPHAT E
The presenc e o f a n enzym e syste m capabl e o f esterifyin g Laglycero phosphate wit h tw o fatt y aci d coenzym e A derivative s wa s first demon -strated i n guine a pi g live r microsome s b y Kornber g an d Price r (62). Th e fatty acid-activatin g syste m wa s als o presen t i n th e sam e preparation ; thus, i t wa s capabl e o f esterifyin g L-a-glycerophosphat e whe n coenzym e A, A T P , an d fatt y acid s wer e adde d t o th e system . Variou s fatt y acid s were esterifie d a t differen t rates , an d i n th e cas e o f palmitat e ther e wa s a suggestion o f inhibitio n b y hig h concentrations . Fluorid e wa s no t in -hibitory, an d althoug h palmitoylhydroxami c aci d wa s no t incorporated , the additio n o f hydrox y lamin e a t 0. 2 M wa s no t inhibitor y an d a t 0. 4 M produced onl y 2 5 % inhibition. Thi s suggest s tha t th e ^-glycerophosphat e esterifying syste m react s muc h mor e rapidl y wit h th e acy l Co A compound s than doe s hydrox y lamine . Cystein e wa s necessary , an d n o requiremen t for M g
+
+ coul d b e demonstrated . Stansl y (68) ha s als o demonstrate d th e need fo r sulfhydry l compound s i n th e system , bu t ther e hav e bee n n o reports o f inhibitio n b y —S H reagents . Th e microsoma l syste m i s thu s able t o brin g togethe r tw o negativel y charge d substrate s an d effec t reac -tion betwee n them . Obviously , th e physicochemica l condi-tion s associate d with thes e reaction s ar e complex , an d a complet e understandin g o f th e system mus t awai t furthe r studie s o f bot h th e fatt y acidactivatin g en -zymes an d th e acylatin g enzym e i n th e natura l lipoprotei n environmen t provided b y th e reticulum . Inhibitio n o f th e fatt y acid-activatin g enzym e will b e discusse d i n othe r chapter s o f th e book .
3. PHOSPHATIDI C ACI D PHOSPHATAS E
The enzym e ha s bee n studie d i n a particulat e preparatio n o f chicke n liver (64), whic h i s fre e o f mitochondri a an d
probabl y
consist s o f micro -somes. I t i s inhibite d b y a rathe r nonspecifi c rang e o f divalen t cations , viz., Ca+ + , M g
+ +
, an d Ba + +
. Thi s ma y b e du e t o maskin g o f th e negativ e charge o f th e phosphatidi c aci d o r eve n t o it s precipitatio n a s a n insolubl e salt. Th e inhibitio n i s mor e marke d i n Tri s buffe r a t p H 7. 4 tha n i n maleat e buffer a t p H 6.3 . Th e author s suggeste d tha t thi s wa s becaus e insolubl e salt formatio n wa s favore d a t th e highe r pH . However , th e effec t coul d also b e du e t o bindin g o f th e divalen t cation s b y th e dicarboxyli c aci d i n the maleat e buffer . Also , a t th e tw o pH' s th e particulat e enzym e prepara -tion ma y exhibi t differen t reactivitie s wit h th e meta l ions . I t wa s als o inhibited b y Twee n 20 , probabl y du e t o disorganizatio n o f th e lipoprotei n structures o f th e particles . Consisten t wit h this , effort s t o prepar e th e enzyme i n a solubl e for m wer e unsuccessful , suggestin g tha t th e structura l integrity o f th e particle s i s necessar y fo r enzym e activity .
200 J. Β. DAVENPORT 4. GLYCERIDE TRANSFERASES
The transfer of phosphorylcholine to diglyceride has been studied with chicken and rat liver mitochondria and microsomes (66), rat brain mito
chondria (66), and rat seminal vesicle mitochondria (67), while phos-phorylethanolamine-glyceride transferase has been demonstrated in a rat liver homogenate (66), and in brain tissue (68). Magnesium ions were necessary for transferase activity (65). Calcium ions and barium ions were inhibitory, as was also fluoride. This striking difference between the effect of M g
+ +
, on one hand, and Ca + +
and Ba+ +, on the other, contrasts with the effect of these ions on the phosphatidic acid phosphatase and suggests a specific role for M g +
+
. This pattern of activation by M g ++
and in
hibition by Ca + +
also resembles that encountered with the glycerokinase and, indeed, most reactions involving transfer of phosphate from nucleo
tides. In some cases (69) it has been shown that Mn+ + can replace M g + +
; but higher concentrations of Mn+ + were inhibitory. In systems to which added cytidine diphosphate choline is converted to lecithin relying on endogenous lipid acceptors, magnesium stimulates activity. This suggests that diglyceride is available in the particulate preparation, as phosphatidic acid phosphatase would be inhibited by the Mg++. However, added di
glyceride emulsified with Tween 20 greatly stimulated lecithin synthesis.
Higher concentrations of Tween 20 (1 mg/ml) were, however, inhibitory, presumably due to some disorganization of the particulate system.
6. PHOSPHOKINASES
The first step in the incorporation of the nitrogen bases into phospho
lipids is their phosphorylation at the expense of A T P . Wittenberg and Romberg (70) partially purified a soluble choline phosphokinase from yeast and tested its activity towards a number of substrates. It phos
phorylated the following substrates in decreasing order of activity: choline, β-dimethylaminoethyl alcohol, β-diethylaminoethyl alcohol, /3-methyl-aminoethyl alcohol, β-ethyl/3-methyl-aminoethyl alcohol, and ethanolamine. There was no activity with serine. Activity appears to fall off with decreasing basicity of the substrate. Cysteine and Mg+ + were necessary for optimum activity. Similar phosphokinase activity was also demonstrated in acetone powders of liver (calf, rabbit, rat, pig), brain (calf and rabbit), intestinal mucosa (calf and rabbit), and kidney (rabbit and pig).
Choline phosphokinase activity has also been demonstrated in one plant tissue, rapeseed (71). The soluble enzyme was partially purified and a thorough study made of its activation and inhibition by a wide range of metal ions. Magnesium was required for activity, but at concentrations above 0.008 M it became inhibitory. Cobalt could partially replace Mg+
+
5. PHOSPHOLIPIDS 201 as an activator, while Mn+ + and Ni+
+
were without effect. M n +
+ was a powerful inhibitor of the Mg+ +-activated enzyme from rapeseed and the yeast and rat-liver enzymes. Optimum activity was always obtained with equimolar concentrations of A T P and Mg+
+
, excess of either resulting in inhibition. A D P inhibited the reaction, presumably by a mass action effect, and this inhibition could not be reversed by the addition of Mg+
+ . The other product of the reaction, phosphorylcholine, also inhibited. A range of heavy metals (Zn++, Al
3
+, Cu++, P g + +
, and Hg++) was in
hibitory, as was also EDTA, presumably due to chelation of the M g
+ +
. In contrast to the animal enzyme, cysteine and glutathione did not activate the enzyme, nor were —SH reagents inhibitory. Diethyl- and dimethylaminoethanols were phosphorylated at slower rates than choline, and the enzyme was without activity towards serine and ethanolamine.
Thus, the reaction appears to involve a stoichiometric combination of the active center of the enzyme, A T P , Mg+ +, and a substrate of high basicity.
Inhibition or reduced activity results from an excess of either A T P or Mg+ +, the substitution of other metal ions for M g
+ +
and accumulation of reaction products.
7. CYTIDYL TRANSFERASES
Borkenhagen and Kennedy studied the phosphorylcholine-cytidyl transferase in a particulate preparation of guinea-pig liver (72). Once again, attempts to solubilize the enzyme were unsuccessful. Heating the preparation for 10-20 min at 55° destroyed phosphorylethanolamine-cytidyl transferase and -glyceride transferase activity while greatly en
hancing · phosphorylcholine-cytidyl transferase activity. Magnesium or manganese ions at a concentration of 0.002 M were required for optimum activity. Higher concentrations of manganese were distinctly inhibitory.
8. "PLASMALOGEN DIGLYCERIDE" TRANSFERASE
The α,β-unsaturated ether analogues of diglyceride and their reaction with cytidine diphosphate choline or ethanolamine to form the corre
sponding plasmalogens has been studied by Kiyasu and Kennedy in a particulate fraction from rat liver (78). The pattern of activation (require
ment for Mg+
+
) and of inhibition (by Ca+
+
) was, not surprisingly, identi
cal with that for the diglyceride transferase. The question of whether the same enzyme is involved in both cases remains to be demonstrated, but the very similar behavior of both systems suggests that it may indeed be the same enzyme. The recent demonstration (74) of naturally occurring plasmalogen analogues of triglycerides suggests that the "plasmalogen diglycerides
,,
may well occur in vivo and be the precursors of the plas
malogens.
202 J. Β. DAVENPORT
9 AND 10. SYNTHESIS OF PHOSPHATIDYLINOSITOL
The synthesis of phosphatidylinositol by the reactions shown in Fig. 6 have been demonstrated by Paulus and Kennedy (75) in liver microsomes.
Manganese appears to be a requirement for both reactions, but no in
hibitory studies have been carried out so far. Reaction (11), which had been postulated by Agranoff et al. (76) to explain the stimulation of labeled inositol incorporation into the monophosphatide by cytidine diphosphate choline, probably does not occur, as Paulus and Kennedy could not demon
strate the release of phosphorylcholine. The latter authors showed that this inositol incorporation was, in fact, an exchange reaction which Ag
ranoff et al. had shown required M g + +
or M n +
+ and the M g + +
-stimulated system to be inhibited by Ca+ +. They made the important comment that, in the presence of M g
+ +
, phosphatidic acid phosphatase activity would be inhibited; thus phosphatidic acid would be available for phosphatidyl-inositol synthesis, and the route via diglyceride to the choline and ethanol
amine phosphoglycerides would be blocked.
(12)
(14)
(15)
Palmityl CoA
/ ^ T P N + CoA ,S*.TPNH Palmitaldehyde
/""Serine
Dihydrosphingosine [/"Flavin js^Flavin H2
Sphingosine
l/~Fatty acyl C o A Js^CoA
JV-Acyl sphingosine (Ceramide)
(16)
^ C D P choline , S^ C M P
Sphingomyelin
F I G . 7. Reactions involved in the biosynthesis of sphingomyelin.
5. PHOSPHOLIPIDS 203
12, 13, AND 14. T H E SYNTHESIS OF SPHINGOSINE
These reactions, synthesizing sphingosine from CoA derivatives of fatty acids and serine, have been demonstrated with rat-brain microsomes (77, 78). In experiments in which the incorporation of palmitaldehyde was studied, the addition of a low concentration of Tween 20 was necessary to disperse the palmitaldehyde. Higher concentrations, however, inhibited the incorporation of serine into sphingosine. Although this suggests that the structural integrity of the particles is necessary for the synthesis, the authors showed that some 2 0 - 3 0 % of the synthetic activity could be re
leased in a soluble form by sonic disintegration of the particles. The addi
tion of D P N or CoA to the incubation medium caused a marked reduction in the conversion of palmitaldehyde to sphingosine; this reduction was probably due to the palmitaldehyde reacting by other pathways involving D P N or CoA.
15. T H E ACYLATION OF SPHINGOSINE
Zabin (79) demonstrated the acylation of sphingosine with a complete system involving the enzymes 12, 13, and 14 as well as the acylation step.
No inhibitory studies have been reported.
16. PHOSPHORYLCHOLINE CERAMIDE TRANSFERASE
The final step in the synthesis of sphingomyelin is formally analogous to the final step in the synthesis of lecithin, the glyceride transferase step.
Sribney and Kennedy (80) showed that both mitochondria and microsomes from chicken liver mediated the reaction. An unusual feature is that the most active ceramide substrate was the N-acetyl-DL-^reo-frans-sphingosine, whereas the corresponding erythro isomer, which is present in all naturally occurring samples of sphingomyelin so far examined, was not incorporated to any extent. The acetylenic iAreo-ceramide with a triple bond in place of the trans double bond was an active substrate but the dihydroceramide and the cis compound were not incorporated. Apparently, the enzyme system required an unsaturated substrate with a configuration about the center of unsaturation provided by either a trans double bond or a triple bond, both of which are very similar. The authors produced evidence that the enzymically synthesized sphingomyelin also had the threo configuration and, furthermore, that enzyme systems isolated from a wide variety of tissues showed the same specificity for the threo-ceramides. The reason for this discrepancy between the substrate specificity of enzyme systems synthesizing sphingomyelin and the configuration of sphingomyelins
204 J. Β. DAVENPORT
isolated from living tissue remains to be clarified. The various ceramide isomers were not tested to see if they could act as competitive inhibitors of the threo isomer, and such investigations would throw further light on the problem.
The ceramide transferase required Mn+
+
for optimum activity, M g +
+ was much less effective, and Ca
+
+ inhibited the system. The shorter-chain acyl ceramides (up to C4) were effective substrates, but longer-chain acyl derivatives (up to Cs) required the presence of Tween 20 before they were incorporated. Once again, higher concentrations of Tween 20 were in
hibitory.
III. ION TRANSPORT AND PHOSPHOLIPID METABOLISM